Microscopy Midterm

下面是我做 teaching fellow 的那門 microscopy 課的 midterm 考題，看了一下出得很不錯。

總分 100 分加上 extra credit 10 分，有興趣的可以試試，我可以幫你改喔，不懂再問助教吧！


1) In class we discussed the light path between the light source and the specimen in a trans-illumination wide-field microscope. You may remember that there are two variable irises in the condenser that modify the light hitting the specimen: the aperture diaphragm and the field diaphragm.

a) What does the aperture diaphragm do to the light? To help explain your answer, draw a ray trace from the filament to the specimen.

b) What does the field diaphragm do? To help explain your answer, draw a ray trace from the filament to the specimen.

In an epi-illumination microscope such as used commonly in fluorescence microscopy, the objective serves double duty both to illuminate the specimen and to image it. In the light path from the arc-lamp to the specimen in an epi-illumination scope there is usually an aperture diaphragm and field diaphragm that serve comparable functions to those in the trans-illumination path.

Let us consider the aperture diaphragm in the epi-illumination path.

c) Draw a ray trace of the light from two different positions on the arc to the specimen plane. You can assume there is a collector lens between the aperture diaphragm and the arc and you can assume for simplicity that the objective is a single lens element. As this is an infinity corrected objective the specimen plane is at the focal distance to the objective. Be sure to show where the back focal plane of the objective is located.

Assume you are looking at a sample that is highly fluorescent with an epi-illumination microscope. For the comfort of your eyes, you decide to reduce the intensity of the fluorescent emission you are looking at. You have two choices: 1- you can close down the aperture diaphragm in the epi-illumination path to a suitable level or 2- you can place a neutral density filter in front of the arc that will remove some portion of the illuminating light. Neutral density (ND) filters are often partially transmissive mirrors that reflect or transmit light- this way they don’t get too hot. They are called neutral because they filter all wavelengths equally. You have a series of ND filters that transmit 1%, 10%, 20%, etc of the light, so you can choose the most suitable one.

d)Does it matter which one you choose? Why or why not?

You next try another sample and find to your dismay that the fluorescence is so faint that you can hardly detect it even with the aperture diaphragm completely open and/or no ND filter in place. You were complaining about the fact that your arc lamp was not bright enough to your uncle, who incidentally owns a car dealership, and he told you he had an old search light in his basement just collecting dust that he used to use to get people to come to his dealership to by shining it in the sky. He is happy to donate it to you along with the power supply and a special heat filter and is sure that it has enough power. The arc lamp you were using is a 100 W Hg with an arc that is only .1 mm in diameter. Your uncle has a Xenon arc that is 10,000W with an arc that is 2 cm in diameter. Being adventurous you decide to take him up on his offer. The area of sample (the field of view) you wish to see is 100 ums diameter.

e) Did you get a brighter image? Why or why not? Ray tracing may help.

Lastly a friend of yours who is doing research in a physics lab offers you a 5 watt green laser that has the right wavelength for exciting your dim sample. Because the beam is collimated (a plane wave) you decide to “scan” your specimen by focusing the beam to a spot on the specimen plane and then moving the stage in a raster pattern and collect the light from each spot with a light detector (a PMT). You of course use the appropriate barrier and dichroic filters to block the exciting light. You then compose an image by taking the PMT output for each spot as a brightness value. To set up the light path you first use a green laser pointer (1 mW). When you compare the results of scanning with 0.1 mW and 5 W you are surprised to find that the fluorescence is not much brighter with the big laser

f) Why?

g) Compare the intensity of a focused 1 mw laser to the intensity of a 100 w arc lamp that illuminates a field of 100 ums diameter. What I want to know is, for a fluorescent molecule at the focus of the 1 mW laser, is it receiving more or less excitation than when illuminated by a 100 W arc. Assume that 30% of the light power of the arc is of the appropriate wavelength to excite the fluorophore and reaches the specimen. The objective is a 40 X 1.3 NA oil. In both cases assume you are using a 546 nm wavelength light.

2) DIC is a powerful technique for generating amplitude contrast out of phase differences. It depends critically on special prisms that alter the light paths of light of different polarizations. Describe the principle and design of a DIC microscope. Feel free to draw but don’t hand in downloaded or Xeroxed diagrams

3) Compare the axial and lateral resolution of these two objectives: a 1.4 NA Oil 60 X and a 1.25 NA water 100X. You are using these lenses to look at a 10 um thick fluorescent sample emitting at 500 nm. Which is the better lens? Why?

4) Extra Credit: Make a pinhole camera and take a picture of some Harvard Icon. Explain why your picture is not perfect (unless it is). Provide a description of the device including relevant measurements.